The effect of serotonin on the hematopoietic stem cells of the bone marrow]

Haemopoietic stem cells content and proliferative activity were studied in the bone marrow of female F1 (CBA x C57Bl6) mice after single (50 mg/kg) and chronic (0.5 mg/kg daily for 7 days) serotonin (S) injections. It is shown that 9-day and 12-day COEs contents in the bone marrow of experimental mice has been increasing for 24 h after single S injection. After chronic S injections twofold increase of 12-day COEs is observed without any increasing of 9-day COEs. Total myelokaryocyte number is increased too. The study of proliferative status by in vitro incubation of bone marrow cells with ARA-C has shown that the numbers of 9-day and 12-day COEs in S-phase have increased both after single and chronic S injections. Possible mechanisms of stimulating effect of S on bone marrow stem cells are discussed.     

混合及是血清在骨髓长期培养中的效应

目的揭示脐血清在骨髓长期培养中的效应,为脐血清的应用提供基础。方法以Dexter培养法,观察混合脐血清(MCBS)、组合细胞因子(CK)在长期骨髓培养中对骨髓单个核细胞(BMMNC)形成鹅卵石造血区(CAFC)、长期培养起始细胞(LTC-IC)、有核细胞(NCC)的生长。结果10例人骨髓,106BMMNC培养5周后,CAFC、LTC-IC分别为37.1±12．4／(ml.well),40．9±10．6/(ml.well),NCC由接种时的106/(ml.well)增至（1．63±0．17）106/(ml.well),加入10％MCBS则可使三者得到明显扩增,但不及组合CK;10％MCBS还能明显提高组合CK对三者的扩增;20％MCBS不能取代骨髓长期培养中的血清和组合CK对三者的扩增。结论MCBS中含有类似GM-CSF、SCF、IL-3、IL-6、EPO等一类能使CAFC、LTC-IC、NCC得到明显扩增的“活性物质”。     

In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow

In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ～1 week.     

Self-renewal capacity and clonal succession of haemopoietic stem cells in long-term bone marrow culture

The CFU-s proliferative potential varied greatly during long-term cultivation. Most of the CFU-s in the cultures were represented by cells with low renewal capacity. Pre-CFU-s cells capable of producing multipotential colonies in methylcellulose, which contained CFU-s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. The findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre-CFU-s, but not the CFU-s themselves, are clonogenic progenitors.     

Renewal of hematopoietic stem cell clones in long-term bone marrow cultures

In long-term cultures of murine bone marrow, clonal succession of hemopoietic cells was observed as measured by karyologic analysis. There were high oscillations in self-renewal of CFUs in the cultures. A close correlation between the CFUmix karyotype and mitotic non-adherent cells in culture (but not between these cell types and CFUs) was revealed.     

Stroma-dependent maintenance of cytokine responsive hematopoietic progenitor cells derived from long-term bone marrow culture

Hematopoietic cells maintained for long periods on primary cultures of bone marrow stromal cells formed cobblestone colonies (Dexter's long-term bone marrow culture, LTBC). These stably maintained hematopoietic cells (for 4 months) were transferred to a coculture on an established spleen stromal cell line (MSS62), and maintained under stromal cell layer, where they retained their invasive ability in the restricted space between the stromal cell layer and culture substratum (DFC culture). DFC contained lineage-negative (Lin-), c-Kit+, Sca-1- cells and spontaneously produced Mac-1+, Gr-1+ cells. DFC could not grow in the absence of MSS62 stromal cells, although, GM-CSF, IL-3, or IL-7 stimulated its growth. Production of granulocyte and monocytic cells was maintained by GM-CSF or IL-3 while it was decreased by IL-7. RT-PCR analysis showed that the IL-7 responsive cell population expressed early lymphoid markers (Ikaros, Pax-5, Oct-2, Rag-1, TdT, IL-7R and Imu), while lacking expression of receptors for G-CSF (G-CSFR) and for M-CSF (M-CSFR), or myeloperoxidase (MPO). These results suggested that DFC simultaneously contained lymphoid-committed progenitors and myeloid-committed progenitors, and that cytokines may expand their responding progenitor cells under the influence of signals provided by the stromal cells. Such a stromal cell-dependent culture system may be useful to analyze the switching mechanism from constitutive to inducible hematopoiesis in vitro.     

Granulocyte colony stimulating factor expands hematopoietic stem cells within the central but not endosteal bone marrow region

Granulocyte colony stimulating factor (G-CSF) is clinically well established for the mobilization of hematopoietic stem cells (HSC). Extensive data on the underlying mechanism of G-CSF induced mobilization is available; however, little is known regarding the functional effect of G-CSF on HSC within the bone marrow (BM). In this study we analyzed the proportion and number of murine HSC in the endosteal and central bone marrow regions after 4 days of G-CSF administration. We demonstrate that the number of HSC, defined as CD150(+)CD48(-)LSK cells (LSKSLAM cells), increased within the central BM region in response to G-CSF, but not within the endosteal BM region. In addition the level of CD150 and CD48 expression also increased on cells isolated from both regions. We further showed that G-CSF mobilized proportionally fewer LSKSLAM compared to LSK cells, mobilized LSKSLAM had colony forming potential and the presence of these cells can be used as a measure for mobilization efficiency. Together we provide evidence that HSC in the BM respond differently to G-CSF and this is dependent on their location. These findings will be valuable in developing new agents which specifically mobilize HSC from the endosteal BM region, which we have previously demonstrated to have significantly greater hematopoietic potential compared to their phenotypically identical counterparts located in other regions of the BM.     

Differentiation characteristics of circulating and bone marrow hematopoietic stem cells and the effect of thymus factors

Circulating hemopoietic stem cells (HSC) considerably differ from bone marrow HSC in active erythroid differentiation. After thymectomy of adult animals the number and differentiation of blood HSC remain unchanged, whereas during the cloning of bone marrow cells, a decrease in the number of granulocytic colonies is revealed. In in-vitro experiments, thymalin does not influence the number or differentiation of circulating HSC. On the contrary, in experiments made in vivo, it dramatically lowers erythroid specialization of blood HSC in thymectomized and sham-operated mice, which is followed by the diminution of the total number of circulating HSC. Differentiation of thymectomized mice bone marrow stem cells is completely normalized after thymalin injection. Sham-operated and thymectomized animals' HSC stimulated by thymalin injection become similar to bone marrow cells of normal mice as regards the trend of differentiation. Thymalin injection is likely to change the bone marrow HSC differentiation profile, thereby preventing the release of the cells with erythroid-oriented differentiation from the bone marrow to blood. The influence of thymalin on HSC is mediated by the environmental component which is present in the bone marrow and absent from the peripheral blood.     

Kinetics of hematopoietic stem cells in the bone marrow of hepatectomized mice

Two-thirds of the liver was removed from (CBA X C57BL/6j) F1 female mice. A significant increase of the number of endogenous colonies count in the spleen of partially hepatectomized mice was observed on the 5-th day after the operation. This increase was not associated with the changes in the number of stem cells in the bone marrow as partial hepatectomy at different times after the operation exerted no effect on the number of colony-forming units (CF1) in the bone marrow.     

The study of mechanisms of radioprotective action of indometofen n hematopoietic stem cells in mouse long-term bone marrow cultures

The influence of indometophen (an analog of tamoxiphen) on the dynamic content and the proliferative activity of CFUs (colony-forming units) and CFU-GM (granulocyto-macrophages precursors) and the level of colony-stimulating factor (GM-CSF) in mouse long-term bone marrow cultures were studied for 4 weeks after administration. Five days after indometophen injection the long-term cultures were exposed to irradiation with a dose of 2 Gy and on the time course of postirradiation recovery haemopoietic precursors cells and dynamic release of GM-CSF in the culture supernatants were examined. The data of this report suggest that the mechanisms responsible for the radioprotective action of indometophen may be associated both with its direct effects on the proliferation and differentiation of hemopoietic cellular precursors and with the stimulation of release of growth-differential factors by hemopoietic microenvironmental elements.     

In vivo osteoinductive effect and in vitro isolation and cultivation bone marrow mesenchymal stem cells

Bone marrow contains cell type termed mesenchymal stem cells (MSC), first recognized in bone marrow by a German pathologist, Julius Cohnheim in 1867. That MSCs have potential to differentiate in vitro in to the various cells lines as osteoblast, chondroblast, myoblast and adipoblast cells lines. Aims of our study were to show in vivo capacity of bone marrow MSC to produce bone in surgically created non critical size mandible defects New Zeland Rabbits, and then in second part of study to isolate in vitro MSC from bone marrow, as potential cell transplantation model in bone regeneration. In vivo study showed new bone detected on 3D CT reconstruction day 30, on all 3 animals non critical size defects, treated with bone marrow MSC exposed to the human Bone Morphogenetic Protein 7 (rhBMP-7). Average values of bone mineral density (BMD), was 530 mg/cm3, on MSC treated animals, and 553 mg/cm3 on control group of 3 animals where non critical size defects were treated with iliac crest autologue bone graft. Activity of the Alkaline Phosphatase enzyme were measurement on 0.5, 14, 21, 30 day and increased activity were detected day 14 on animals treated with bone marrow MSCs compared with day 30 on iliac crest treated animals. That results indicates strong osteoinduction activity of the experimental bone marrow MSCs models exposed to the rhBMP-7 factor Comparing ALP activity, that model showed superiorly results than control group. That result initiates us in opinion that MSCs alone should be alternative for the autolologue bone transplantation and in vitro study we isolated singles MSCs from the bone marrow of rat's tibia and femora and cultivated according to the method of Maniatopoulos et all. The small initial colonies of fibroblast like cells were photo-documented after 2 days of primary culture. Such isolated and cultivated MSCs in future studies will be exposed to the growth factors to differentiate in osteoblast and indicate their clinically potential as alternative for conventional medicine and autologue bone transplantation. That new horizons have potential to minimize surgery and patient donor morbidity, with more success treatment in bone regenerative and metabolism diseases.     

Microencapsulated human bone marrow cultures: a potential culture system for the clonal outgrowth of hematopoietic progenitor cells

Currently the most successful methods for culturing human hematopoietic cells employ some form of perfused bioreactor system. However, these systems do not permit the clonal outgrowth of single progenitor cells. Therefore, we have investigated the use of alginate-poly-L-lysine microencapsulation of human bone marrow, combined with rapid medium exchange, as a system that may overcome this limitation for the purpose of studying the kinetics of progenitor cell growth. We report that a 12 to 24-fold multilineage expansion of adult human bone marow cells was achieved in about 16 to 19 days with this system and that visually identifiable colonies within the capsules were responsible for the increase in cell number. The colonies that represented the majority of cell growth originated from cells that appeared to be present in a frequency of about 1 in 4000 in the encapsulated cell population. These colonies were predominantly granulocytic and contained greater than 40,000 cells each. Large erythroid colonies were also present in the capsules, and they often contained over 10,000 cells each. Time profiles of the erythroid progenitor cell density over time were obtained. Burst-forming units erythroid (BFU-E) peaked around day 5, and the number of morphologically identifiable erythroid cells (erythroblasts through reticulocytes) peaked on day 12. We also report the existence of a critical inoculum density and how growth was improved with the use of conditioned medium derived from a microcapsule culture initiated above the critical inoculum density. Taken together, these results suggest that microencapsulation of human hematopoietic cells allows for outgrowth of progenitor, and possible preprogenitor, cells and could serve as a novel culture system for monitoring the growth and differentiation kinetics of these cells.     

Endosteal cells of the bone marrow stroma in human iliac bone

The data on histological and electron microscopical investigation of the endosteal cells of the human iliac bone are presented. Three types of stromal elements in the endosteum, differing in their ultrastructural organization have been revealed. In the endosteal areas young hemopoietic cells are present, they are closely connected with the stroma. A suggestion is made on an important role of the endosteum in processes of proliferation and differentiation of hemopoietic predecessors.     

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

This paper describes a study of hematopoiesis in parathion-treated mice. Adult mice (48 C57B1/6) were given a daily dose of parathion (4 mg/kg p.o.) or corn oil vehicle (5 ml/kg p.o.) for 14 days. During the pesticide and the examination period, treated animals showed no signs of poisoning and had normal body weights. On days 2, 5, 7, 9, 12 and 14 following parathion or corn oil, femoral marrow cells were assayed in vitro for granulocyte/monocyte (CFU-gm), erythroid (CFU-e and BFU-e), megakaryocyte (CFU-meg), stromal (CFU-str) and multipotential (CFU-mix) hematopoietic stem cells. Leukocyte counts were elevated on days 2 and 5, while platelet counts were not increased until day 12. No change was observed in either hematocrits or numbers of marrow cells. BFU-e were reduced (23% of control) by day 7, then increased to 137% of control by day 14. CFU-e were reduced (41% of control) on day 9, then increased to 71% of control by day 14. CFU-mix were 130% of control (day 2), then declined to control values by day 5. On days 12 and 14, CFU-mix colonies decreased to 40% of control. CFU-str were reduced at all time points examined. CFU-gm were 123%, 136% and 130% of control on days 7, 12 and 14, respectively, while CFU-meg were increased (145% of control) on day 7. The data suggest that parathion alters the cloning potential of bone marrow precursor stem cells.     

Function and malfunction of hematopoietic stem cells in primary bone marrow failure syndromes

Hematopoietic stem cells (HSCs) are responsible for the production of mature blood cells in bone marrow; peripheral pancytopenia is a common clinical presentation resulting from several different conditions, including hematological or extra-hematological diseases (mostly cancers) affecting the marrow function, as well as primary failure of hematopoiesis. Primary bone marrow failure syndromes are a heterogeneous group of diseases with specific pathogenic mechanisms, which share a profound impairment of the hematopoietic stem cell pool resulting in global or selective marrow aplasia. Constitutional marrow failure syndromes are conditions caused by intrinsic defects of HSCs; they are due to inherited germline mutations accounting for specific phenotypes, and often involve also organs and systems other than hematopoiesis. By contrast, in acquired marrow failure syndromes hematopoietic stem cells are thought to be intrinsically normal, but subjected to an extrinsic damage affecting their hematopoietic function. Direct toxicity by chemicals or radiation, as well as association with viruses and other infectious agents, can be sometimes demonstrated. In idiopathic Aplastic Anemia (AA) immunological mechanisms play a pivotal role in damaging the hematopoietic compartment, resulting in a depletion of the hematopoietic stem cell pool. Clinical and experimental evidences support the presence of a T cell-mediated immune attack, as confirmed by clonally expanded lymphocytes, even if the target antigens are still undefined. However, this simple model has to be integrated with recent data showing that, even in presence of an extrinsic damage, preexisting mutations or polymorphisms of genes may constitute a genetic propensity to develop marrow failure. Other recent data suggest that similar antigen-driven immune mechanisms may be involved in marrow failure associated with lymphoproliferative or autoimmune disorders characterized by clonal expansion of T lymphocytes, such as Large Granular Lymphocyte leukemia. In this wide spectrum, a unique and intriguing condition is Paroxysmal Nocturnal Hemoglobinuria (PNH); even in presence of a somatic mutation of the PIG-A gene carried by one or more HSCs and their progeny, the typical marrow failure in PNH is likely due to pathogenic mechanisms similar to those involved in AA, and not to the intrinsic abnormality conferred to the clonal population by the PIG-A mutation. The study of hematopoietic stem cell function in marrow failure syndromes provides hints for specific molecular pathways disturbed in many diseases of hematopoietic and non-hematopoietic stem cells. Beyond the specific interest of investigators involved in the field of these rare diseases, marrow failure syndromes represent a model that provides intriguing insight into quantity and function of normal hematopoietic stem cells, improving our knowledge on stem cell biology.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.